Team:Goettingen/week19-2

V09_03

• Experiment:
  In order to eliminate undesired restriction sites inside of our fliC part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and PfuTurbo polymerase (protocol). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained.
  
• Observations & Results:
  The QC PCR was not successful. We were not able to observe any PCR product.

V09_04

• Experiment:
  The test digestion of pSB1C3 including flHDC under the expression of 8 different promoters was performed with the restriction enzymes EcoRI and PstI according to the protocol. The product was subsequently analyzed on an 1% analytical agarose gel.
  
• Observations & Results:
  Since fragments of the expected size could be obtained for all clones the test digestion worked for all.

• Experiment:
  PCR of motA, motB and yhjH using primers containing the standard BioBrick sequences. Procedure was succesful and the PCR products were purified via PeqGOLD Gelextraction Kit (Peqlab).

V09_05

• Experiment:
  motA, motB, yhjH and pSB1C3 were digested with EcoRI and PstI. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).

• Experiment:
  Here again the Overlapping PCR was performed using designed primers and PfuTurbo polymerase (protocol). However, this time a lower annealing temperature was chosen. Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment.
  
• Observations & Results:
  Once again the QC PCR failed for no PCR product was observed. Since we already had problems with enzymes during our experiments we consider an non-functionable polymerase.

V09_06

• Experiment:
  The ligation of motA, motB and yhjH with pSB1C3 was conducted as described in the protocol.
  

• Experiment:
  The transformation was performed as described in the standard protocol.
  
• Observations & Results:
  The transformation was successful since numerous colonies has formed whereas the negative control remained clear.

V09_09

• Experiment:
  Over night cultures were prepared:
  
  pSB1C3-motA
  pSB1C3-motB
  pSB1C3-yhjH